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. Author manuscript; available in PMC: 2018 Nov 20.
Published in final edited form as: J Autoimmun. 2015 Jul 2;62:81–92. doi: 10.1016/j.jaut.2015.06.003

Fig. 3.

Fig. 3.

Repeated apoptotic cell injections induce a GC response in WT, WASp−/− and WASp−/−N-WASpfl/flCD19Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220+ area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp−/− d 0 n = 7, WASp−/− d 27 n = 17, WASp−/−N-WASpfl/flCD19Cre/+ d 0 n = 8, WASp−/−N-WASpfl/flCD19Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp−/− n = 13, WASp−/−N-WASpfl/flCD19Cre/+ n = 11. (C) Absolute number of TFH cells (CD4+CD44+CD62L−PD1+CXCR5+) analyzed by flow cytometry. WT n = 8, WASp−/− n = 7, WASp−/−N-WASpfl/flCD19Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83+CXCR4−) and DZ B cells (CD83−CXCR4+). WT n = 14, WASp−/− n = 13, WASp−/−N-WASpfl/flCD19Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35+ area of total PNA+ or B220+ area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220−CD138+) and IgG1+ plasma cells as determined by flow cytometry. WT n = 8, WASp−/− n = 7, WASp−/−N-WASpfl/flCD19Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp−/− d 0 n = 4, WASp−/− d 27 n = 6, WASp−/−N-WASpfl/flCD19Cre/+ d 0 n = 4, WASp−/−N-WASpfl/flCD19Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. **P < 0.01, ***P < 0.001 and ****P < 0.0001. Outliers based on ROUT (Q = 1%) excluded: (d) 1 WKO. Abbreviations: WKO; WASp−/−, cDKO; WASp−/−N-WASpfl/flCD19Cre/+.